Assay utilizing magnetic particles

ABSTRACT

An assay for the presence of an analyte or determining a biological or medical parameter comprising the steps of adding an assay component to a pH-sensitive material charged with magnetic particles, wherein the assay component causes a pH change which is a function of the analyte or parameter to be measured; subjecting the magnetic particles to an oscillating magnetic field; and measuring the effect of the magnetic field on the magnetic particles. The assay is an enzyme immunoassay. A kit for practicing the enzyme immunoassay is also described.

The present invention relates to assay for the pressure of an analyte orfor determining a biological or medical parameter and to a kit for suchan assay.

When a patient is treated by a physician, it is not uncommon for thephysician to take samples of body fluids to be sent on to a laboratoryfor analysis. Such testing inevitably gives rise to some delay in theprocessing of the sample. This is particularly true where samples haveto be sent to a separate laboratory for testing. Even in hospitals itcan often take a number of hours for the results to be communicated backto the physician. Accordingly, it is not uncommon for the physician tobegin treating a patient without knowing the results of any requestedtesting.

It situations where the patient is seriously ill, the delay incurred intesting samples could conceivably put the well-being of that patient atrisk.

One might consider that a suitable way to overcome this problem would befor the physician in charge of a particular patient to conduct thetesting himself, without sending the samples away to a laboratory.However, the testing of samples is often a complex process which must becarried out by highly trained personnel if the results are to bereliable and hence of any real use to the physician.

Therefore, there is a need in the art for assays which can be quicklyand reliably carried out by a user (who will sometimes be referred to asin operator), particularly for samples obtained from patients.

GB-A-2,284,890 relates to an analyte sensor in which an analyte isdetected by reacting it with an enzyme or catalytic species at or verynear to a polymer coated electrode. The enzyme or catalytic speciesdirectly or indirectly effects a reaction with said polymer layerwherein the polymer layer becomes porous causing a measurably change inelectrical properties at the electrode surface. In one embodiment thepolymer is pH-sensitive and the reactions cause a change in pH.

WO98/10788 relates a method for the measurement of clot formation times,clot dissolution time or clotting parameters by monitoring movement ofmagnetic particles in the sample being assayed, where the movement isinduced by a magnetic field.

WO94/19690 relates to a method for performing an affinity assay andwhich monitors the response of an oscillating field on magneticparticles to determine the analyte concentration.

WO95/06868 also describes an oscillating particle type coagulationassay.

The present invention provides a method of testing samples, especiallypatient samples. Advantages of this method include the fact that theassay can use whole blood, hence avoiding a pre-assay step involvingseparation of blood components. The method of the present invention canbe automated and will make the results of the test available quickly andthus provide an early and rapid diagnosis of a patient's condition. Themethod is simply and therefore minimal operator skill is required. Inaddition, the equipment necessary to automate the method may be readilyavailable and therefore the method will be economic.

In its broadest aspect the present invention provides a method forcarrying out an assay which has as its end point a pH change.

In a first aspect, the present invention provides an assay for thepresence of an analyte or determining a biological or medical parametercomprising the following steps:

an assay component is added to a pH-sensitive material charged withmagnetic particles, wherein the assay component causes a pH change whichis a function of the parameter to be measured;

subjecting the magnetic particles to an oscillating magnetic field; andmeasuring the effect of the magnetic field on the magnetic particles.

The presence of the analyte is determined, or determination of theparameter is made, using analysis of the response of the magneticparticles to the magnetic field.

It will be appreciated that the pH material is changed as a function ofthe analyte or parameter and this results in a change in the movement ofthe magnetic particles in the oscillating magnetic field.

The sample may be aqueous, whole blood, serum, plasma, urine or saliva.

In a second aspect, there is provided a method for carrying out an assayfor the presence of an analyte, comprising:

contacting a sample to be assayed for the presence of an analyte with areactive species, the analyte reacting with the reactive speciesresulting in a change in pH;

directly or indirectly effecting a reaction with a pH-sensitivematerial, which is charged with pH-sensitive material magneticparticles;

applying a magnetic field to the magnetic particles;

monitoring a response of said magnetic particles to the magnetic field;and

determining the amount of analyte in said sample by analysis of theresponse of the magnetic particles.

The change in pH is a function of the presence of the analyte.

The interaction of the analyte with the reactive species gives rise toan assay component and the assay component results in the change in pH.

The analyte and reactive species interact to form an assay componentsuch that the pH change is a function of the presence of the analyte.This function may be allow a qualitative or quantitative determinationof the amount of analyte present in the sample.

Preferably the assay employs binding pairs. A non-exclusive list ofcommonly used binding pairs includes avidin/biotin, antibody/antigen,haptens and nucleic acid (DNA and RNA). Generally when the binding pairis antibody/antigen the assay is referred to as an immunoassay. Otherbiosubstances capable of molecular recognition include lectins forsaccarides, hormone receptors for hormones and drug receptors for drugsand active drug metabolites.

In a preferred aspect the method is used for performing an immunoassay.

In a third aspect, the present invention relates to an enzymeimmunoassay in which the pH produced by the enzyme reaction is afunction of the amount of analyte, and wherein liberation of magneticparticles from a pH-sensitive material is detected.

As changes in pH are associated with many enzyme reactions the presentinvention provides a flexible assay with wide applicability. Generally,in enzyme immunoassays, the enzyme is used as a label or marker which isbound to one member of the antigen-antibody pair identical to that inthe sample to be measured. The enzyme bound antigen/antibody thencompetes with the sample antigen/antibody for the binding site on alimited supply of its complement antibody/antigen.

Classical methods for immunoassay include:

(i) A capture antibody on a solid phase, such as a plastic microtitreplate, exposure to the biological sample to attach the antigen ofinterest, washing and then exposure to a second labelled antibody. Thelabel on the antibody may be an enzyme for example. Further washing isfollowed by detection of the label (and hence the amount of antigen inthe original sample). This is known as a sandwich assay or two-siteassay, or

(ii) A capture antibody on the solid phase followed by exposure to thebiological sample containing antigen and an added amount of labelledantigen. Labelled and unlabelled antigen compete on the solid phase forthe antibody sites. The amount of label revealed after washing isinversely proportional to the amount of true antigen in the biologicalsample. This is known as a competitive assay.

Other immunoassays methods which are known, or become known, to askilled worker may also be used. For example, the assay may use directmolecular recognition. In this approach, one of the binding pair, e.g.an antibody, which is immobilised binds with its binding pair present inthe sample, causing a pH change. The advantage of direct-recognitionassays is simplicity.

In enzyme immunoassays the label is measured by adding the enzymesubstrate and monitoring the product by a suitable method. In thepresent case, the enzyme reaction gives rise to a pH change and thismonitored using the effect of a magnetic field on magnetic particlescaptured in a pH-sensitive material. The signal is then processed and aresult calculated.

In another preferred aspect the method of the present invention is usedfor performing an assay for biological or medical parameters.

Thus, according to a fourth aspect of the present invention there isprovided a method for carrying out an assay for biological and medicalparameters, comprising:

contacting a sample to be assayed for a biological or medical parameterwith a reactive species causing an assay component to be developed whichis a function of the parameter to be measured and resulting in a changein pH;

directly or indirectly effecting a reaction with a pH-sensitive materialwhich is charged with magnetic particles;

applying a magnetic field to the magnetic particles;

monitoring a response of said magnetic particles to the magnetic field;and

determining the parameter by analysis of the response of the magneticparticles.

The change in pH is a function of the biological or medical parameter.

The interaction of the sample with the reactive species gives rise to anassay component and the assay component results in the change in pH.

The sample and reactive species interact to form an assay component,which results in a pH change which is a function of the parameter to bedetermined. This function may be allow a qualitative or quantitativedetermination of the parameter of interest.

The biological or medical parameters may include clinical chemistryparameters, typically blood electrolytes and metabolites, or blood gasmeasurement.

In a preferred embodiment, particularly for clinical chemistry or bloodgas assays, the sample is exposed to a combination of reagents, normallyenzymes and substrates, which cause an assay component to be developedin proportion to the presence of the analyte of interest. Thus,according to another aspect of the present invention there is providedan assay for biological or medical parameters, such as clinicalchemistry or blood gas measurements, in which a sample containing ananalyte mixes with reagents suitable to cause a pH change in proportionto the analyte concentration. It will be appreciated that in this casethe analyte corresponds to the parameter of interest.

The method of the present invention is described below mainly inconnection with immunoassays; however, it will be appreciated that thegeneral description is equally applicable to other assays such as themeasurement of biological or medical parameters.

The amount or presence of analyte may be determined by analysis of thetime varying signal of the magnetic particles as they are subjected tothe oscillating magnetic field.

The magnetic field may be generated by the use of a static or movingmagnetic field. Preferably the oscillating field is generated by anoscillating field generating device which is itself stationary.

In a preferred embodiment the assay uses a small chamber containing themagnetic particles captured in the pH-sensitive material. The assaycomponent is added to the chamber. The magnetic particles are subject toan oscillating magnetic field. This movement is detected by monitoringthe light reflected from a source shown down onto the chamber. As theparticles stand up reflected light from the card is reflected onto aphotodetector placed above the chamber. As the particles lie down thelight is blocked and a minimum is reached on the photodetector. As thepH-sensitive material dissolves or disintegrates the particles becomefreed to move and the level of light reflected changes, preferably in aspecific time dependent way.

Preferably the chamber forms part of a plastic card system.

The polymer is preferably in the form of a thin film and is generallyalmost translucent. Enteric polymers can be selected which have theproperty of dissolving at a specific pH in solution.

An assay can be designed in wicking format which creates a pH change ina suitable buffer solution or in the patient sample itself. The pHchanges will be dependent upon the quantity of analyte present, and thiscauses the polymer to dissolve and the particles to move. The movementof the particles would be detected as a change in reflected lightlevels. The time dependent behaviour of the reflected light would beused to calibrate the immunoassay.

Conveniently the magnetic particles are fixed in the pH-sensitivematerial by cross-linking the molecules of the pH-sensitive materialthereby trapping or capturing the magnetic particles.

The present invention involves the partial or complete removal of thepH-sensitive material from around the magnetic particles. As previouslymentioned, preferably the present invention relates to an enzymaticimmunoassay. Generally in this embodiment, the enzyme or catalystproduces a product which can react with the polymer, or can directlyhydrolyse the polymer membrane.

The enzymes or catalysts may be bound within or directly to the polymere.g. bound to the polymer via an antibody-antigen interaction with anenzyme-labelled conjugate, bound to a porous membrane in close proximityto the pH-sensitive material, or be present in the bulk solution.Preferably, the enzymes or catalysts will be contained in a secondreaction chamber, the product of the enzyme reaction (also known in thepresent invention as the assay component) then entering the chambercontaining the pH-sensitive material and magnetic particles.

One of the many appropriate combinations of an enzyme with a polymercoating is the combination of urease with materials such as those usedin enteric coatings for tablets. These coating work by being insolubleat the low pH of the stomach, but are soluble at the pH in the intestine(pH 6 and above). Examples of such coating materials are celluloseacetate hydrogen phthalate, methyl vinyl ether-maleic anhydridecopolymer esters, anionic polymerisates of methacrylic acid and estersof methacrylic acid (Eurragit® of Rohm-Pharma, Darmstadt, Germany).

In one particular reaction, urease catalyses the breakdown of urea toammonia and carbon dioxide according to the following scheme:

 H₂O+urea→2NH₃+CO₂

NH₃+H₂O→NH₄ ⁺+OH⁻

The resulting increase of the pH value leads to a solubilization of thepolymer.

If an antibody is immobilized onto said pH-sensitive polymers, it maycapture an urease-labelled conjugate in an immunoassay. The bound ureaseconjugate then produces a local pH change that leads to thesolubilisation of the polymer at the point of conjugate capture. We havefound that local solubilisation can occur in solutions where, because ofthe buffering capacity of the bulk solution, a significant pH change inthe solution does not take place. Since assays according to thisinvention do not require a wash step they can fulfil all therequirements of homogenous assays.

In another embodiment, it is possible to use an H₂O₂-producing enzymesuch as glucose oxidase in conjunction with Fenton's reaction. Thisreaction is commonly used in synthetic organic chemistry to hydroxylatearomatics, and works by producing the extremely reactive radical OH;e.g.

H₂O₂+Fe²⁺→Fe³⁺+OH⁻+HO

The hydroxyl radical is so reactive that it survives only until itencounters an organic species. In this case a polymer coating is chosenwhich contains structural elements reacting with the HO radicals. Theintroduction of hydroxyl groups enhances the solubility of the coating.

The assay according to the present invention may be carried out in manydifferent formats known to the skilled worker.

So it is possible to immobilise in any manner known to the man skilledin the art, an antibody at the polymer surface, or preferably in asecond reaction chamber, for which the analyte to be measured and ananalyte enzyme conjugate or an analyte analogue enzyme conjugatecompete.

For a sandwich format immunoassay a first antibody against the analyteto be measured is immobilised at the pH-sensitive material and a secondantibody labelled with an enzyme is present in the solution. The firstantibody binds the antigen (analyte) of interest in the sample. Thesecond antibody, labelled with an enzyme, binds to the captured antigen.A substrate is present which is converted by the enzyme to the assaycomponent causing a pH change.

For a competition format, a capture antibody is immobilised on a solidphase and then a sample is added containing labelled and unlabelledantigen which compete the antibody sites on the solid phase. The amountof label revealed is inversely proportioned to the amount of antigen inthe sample.

In one embodiment of a competition format the solution contains a biotinlabelled analyte or a biotin labelled analogue of the analyte. An enzymeconjugate with avidin is also present in the solution or may be addedafter the capture reaction of the biotin labelled analyte or analyteanalogue and the analyte to be measured with the immobilised antibody.Other binding pairs instead of avidin/biotin, e.g. IgG:anti-IgG may beused equally.

As previously mentioned, it is also possible to use a competitive assaywhere an analyte or an analogue of the analyte is conjugated with ananti-enzyme antibody. Also present in solution is the analyte to bemeasured and free enzyme, where the signal generated is inverselyproportional to the analyte concentration being measured.

In a preferred format, the interaction between the analyte and an enzymeconjugate of the analyte or a analogue of the analyte with an enzyme canbe performed in a wick (bibulous layer) or a capillary channel in whichan antibody against the analyte is immobilized on the surface. Afterhaving passed the wick or capillary channel, the unbound enzymeconjugate of the analyte comes into contact with the material where ananti-enzyme antibody is immobilised. The signal generated isproportional to the concentration of analyte present.

In a further preferred embodiment, the reagent systems may be designedto allow clinical chemical parameters, including blood gas measurement,to be carried out on the same instrument.

Various preferred features and embodiments of the present invention willnow be described by way of non-limiting example, with reference to theaccompanying drawing, in which:

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows a schematic layout of an assay in accordance with oneembodiment of the present invention suitable for detecting an analyte;

FIG. 2 shows a schematic layout of an assay in accordance with anotherembodiment of the present invention suitable for the analysis ofparameters of biological or medical interest; and

FIG. 3 shows a schematic layout of a method of detection in accordancewith one embodiment of the present invention.

EXAMPLE 1

This assay is described with reference to FIG. 1.

Steps:

a) Whole blood is added over blood separation membrane 1. The plasmaflows along channel to area 2.

b) Antigen picks up tracer antibody at area 2. The tracer antibody islabelled with an enzyme which can cause a pH change when in the presenceof its corresponding substrate (e.g. urease).

c) Capture antibody at area 3 binds antigen-labelled antibody conjugate.Excess sample and tracer antibody is drawn off by a wicking action intoabsorbent area 4.

d) An enzyme substrate (e.g. urea) is released from urea 5 and washesover area 3 causing a pH change when it contacts the enzyme label.

e) A solution with a pH which varies over time arrives at pH-sensitivematerial and magnetic particle mix in chamber 6.

f) The pH-sensitive material begins to dissolve in a time-dependentmanner which is a function of antigen concentration. The magneticparticle are able to move in an applied oscillating magnetic field andcause changes in reflected light which are detected.

The reagent sample flow between areas 4, 5 and 6 may be controlled byany convenient means, such as pressure contact changes to control flowarea and open substrate material.

EXAMPLE 2

This assay is described with reference to FIG. 2.

a) Whole blood is added over sample chamber 1. In one embodiment samplechamber 1 contains a blood separation membrane. In another embodiment,it is simply a chamber for introducing sample to the diagnostic testcard.

b) Whole blood or plasma is channelled by wicking, or flows along thecard, to reaction area 2. The sample mixes with the reagents in area 2which are selected to as to cause a pH change related to the parameterto be measured.

c) The reacted liquid flows, or is drawn by wicking, into chamber 3which contains the pH-sensitive material and magnetic particle mix.

d) The pH-sensitive material begins to dissolve in a time dependentmanner which is a function of the concentration of the parameter beingmeasured. The magnetic particles are able to move in an appliedoscillating magnetic field and cause changes in reflected light whichare detected.

It will be appreciated that the above tests can be carried out on a testcard, which is preferably about the size of a credit card, having areaction chamber containing paramagnetic iron oxide particles capturedin the pH-sensitive material.

In order to run a test a sample drop is added to the card in anappropriate well, which could be the chamber itself containing themagnetic particles and pH-sensitive material of a separate reactionchamber. In the present Examples the sample drop would be added to area1 as shown in FIGS. 1 and 2.

The photodetector detects any change in the light from the added sample.While the test is in progress an electromagnet turns on and off everysecond. As the paramagnetic particles in the test card became freed theystand up when the magnet is on, causing more light to pass to thephotodetector. When the magnetic is off, the particles fall down causingless light to be detected. The movement of these particles produces thesignal. Disintegration of the pH-sensitive materials causes theparticles to start movement. The analyser monitors particle movement.

As illustrated in FIG. 3, an optical sensor 1 produces light which isdirected to a sample 2 on a test card 3. The sample has no or atransparent cover. Light from the source reflects off a reflectingsurface beneath the cover. The reflected rays pass into a photodetector4. An electromagnetic which generates the oscillating magnetic field isshown at 5.

What is claimed is:
 1. An assay for the presence of an analyte ordetermining a biological or medical parameter comprising: adding anassay component to a pH-sensitive material charged with magneticparticles, wherein the assay component causes a pH change which is afunction of the analyte or parameter to be measured; subjecting themagnetic particles to an oscillating magnetic field; and measuring theeffect of the magnetic field on the magnetic particles.
 2. The assayaccording to claim 1 wherein the assay component results from theinteraction of the sample to be assayed with a reactive species.
 3. Anassay for the presence of an analyte, comprising: contacting a sample tobe assayed for the presence of an analyte with a reactive species, theanalyte interacting with the reactive species resulting in a change inpH; directly or indirectly effecting a reaction with a pH-sensitivematerial which is charged with magnetic particles; applying a magneticfield to the magnetic particles; monitoring a response of said magneticparticles to the magnetic field; and determining the amount of analytein said sample by analysis of the response of the magnetic particles. 4.An assay for determining a biological or medical parameter, comprising:contacting a sample to be assayed for a biological or medical parameterwith a reactive species, the sample interacting with the reactivespecies resulting in a change in pH; directly or indirectly effecting areaction with a pH-sensitive material which is charged with magneticparticles; applying a magnetic field to the magnetic particles;monitoring a response of said magnetic particles to the magnetic field;and determining the parameter by analysis of the response of themagnetic particles.
 5. The assay according to claim 2 wherein thereactive species is urease or glucose oxidase.
 6. The assay according toclaim 1 wherein the assay component is a hydroxyl radical.
 7. The assayaccording to claim 1 wherein the pH-sensitive material is an entericpolymer, acetate hydrogen phthalate, a methyl vinyl ether-maleicanhydride ester, an anionic polymerisate of or an ester of.
 8. The assayaccording to claim 1 wherein the assay is an immunoassay.
 9. A kit forperforming the assay of any of claims 1-8 comprising a reaction chambercontaining magnetic particles captured in an pH-sensitive material,means for generating a magnetic field and detection means.
 10. A bindingpair assay in which a binding pair reaction produces a pH which is afunction of the presence of an analyte in a sample or biological ormedical parameter of a sample, and which comprises detecting liberationof magnetic particles from a pH-sensitive material.
 11. The assayaccording to claim 3 wherein the reactive species is urease or glucoseoxidase.
 12. The assay according to claim 4 wherein the reactive speciesis urease or glucose oxidase.
 13. The assay according to claim 5 whereinthe reactive species is urease or glucose oxidase.
 14. The assayaccording to claim 7 wherein the pH-sensitive material is an entericpolymer selected from the group consisting of cellulose acetate hydrogenphthalate, a methyl vinyl ether-maleic anhydride ester, an anionicpolymerisate of methacrylic acid and an ester of methacrylic acid. 15.The assay according to claim 1 wherein the assay is an enzymeimmunoassay.